Modified vaccinia virus and methods for making and using the same

ABSTRACT

What are disclosed are methods for modifying the genome of vaccinia virus to produce vaccinia mutants, particularly by the introduction into the vaccinia genome of exogenous DNA; modified vaccinia prepared by such methods; certain DNA sequences and unmodified and genetically modified microorganisms involved as intermediates in such methods; and methods for infecting cells and host animals with such vaccinia mutants to provoke the amplification of exogenous DNA and proteins encoded by the exogenous DNA, including antigenic proteins, by said cells and host animals.

The invention described herein was made with the support of the FederalGovernment and the Federal Government has certain rights in theinvention.

The present invention relates to modified vaccinia virus, to methods ofmaking and using the same, and to other modified and unmodifiedmicroorganisms, and to certain DNA sequences, produced or involved asintermediates in the production of modified vaccinia virus. More inparticular, the invention relates to vaccinia virus in which thenaturally occurring genome of the virus has been altered ("vacciniamutants") and to methods of making and using such vaccinia mutants, aswell as to other unmodified and genetically modified microorganisms, andto certain DNA sequences, produced or involved as intermediates in theproduction of vaccinia mutants.

Vaccinia virus is the prototypic virus of the pox virus family and, likeother members of the pox virus group, is distinguished by its large sizeand complexity. The DNA of vaccinia virus is similarly large andcomplex. Vaccinia DNA is about 120 megadaltons in size, for instance,compared with a DNA size of only 3.6 megadaltons for simian virus 40(SV40). The DNA molecule of vaccinia is double-stranded and terminallycrosslinked so that a single stranded circle is formed upon denaturationof the DNA. Vaccinia DNA has been physically mapped using a number ofdifferent restriction enzymes and a number of such maps are presented inan article by Panicali et al., J. Virol. 37, 1000-1010 (1981) whichreports the existence of two major DNA variants of the WR strain ofvaccinia virus (ATCC No. VR 119) which strain has been most widely usedfor the investigation and characterization of pox viruses. The twovariants differ in that the S("small") variant (ATCC No. VR 2034) has a6.3 megadalton deletion not occurring in the DNA of the L("large")variant (ATCC No. VR 2035). Maps obtained by treatment of the variantswith the restriction enzymes Hind III, Ava I, Xho I, Sst I, and Sma Iare presented in the aforementioned article.

Vaccinia, a eukaryotic virus, reproduces entirely within the cytoplasmof a host cell. It is a lytic virus, i.e. a virus, the replication ofwhich in a cell results in lysis of the cell. The virus is considerednon-oncogenic. The virus has been used for approximately 200 years invaccines for inoculation against smallpox and the medical profession iswell acquainted with the properties of the virus when used in a vaccine.Although inoculation with vaccinia is not without risk, the risks are onthe whole well known and well defined and the virus is consideredrelatively benign.

At the heart of the present invention is the modification of thenaturally occurring vaccinia genome to produce vaccinia mutants byrearrangement of the natural genome, by the removal of DNA from thegenome, and/or by the introduction into the naturally occurring vacciniagenome of DNA which disrupts the naturally occurring genome ("foreignDNA"). Such foreign DNA may be naturally occurring in vaccinia or may besynthetic or may be naturally occurring in an organism other thanvaccinia. If genetic information is present in this foreign DNA, thepotential exists for the introduction of this information into aeukaryote via modified vaccinia virus. That is, the modified virusrepresents a relatively innocuous eukaryotic cloning vector from whichgenetic information has been deleted, or into which information has beeninserted or in which genetic information has been rearranged. Since thevirus replicates within the cytoplasm of an infected cell modifiedvaccinia virus represents a unique eukaryotic cloning vector unlike anyother so far considered or currently under investigation.

This discovery has a number of useful consequences, among which are (A)novel methods for vaccinating mammals susceptible to vaccinia to inducein them an antibody response to antigens coded for by foreign DNAinserted into the vaccinia virus, (B) novel methods for the productionby eukaryotic cells of biological products other than antigens, and (C)novel methods for the introduction into human or animal individuals orpopulations of missing genes or of genetic material for themodification, replacement, or repair of defective genes in theindividuals or populations.

Suitably modified vaccinia mutants carrying exogenous genes which areexpressed in a host as an antigenic determinant eliciting the productionby the host of antibodies to the antigen represent novel vaccines whichavoid the drawbacks of conventional vaccines employing killed orattenuated live organisms. Thus, for instance the production of vaccinesfrom killed organisms requires the growth of large quantities of theorganisms followed by a treatment which will selectively destroy theirinfectivity without affecting their antigenicity. On the other hand,vaccines containing attenuated live organisms always present thepossibility of a reversion of the attenuated organism to a pathogenicstate. In contrast, when a modified vaccinia mutant suitably modifiedwith a gene coding for an antigenic determinant of a disease-producingorganism is used as a vaccine, the possibility of reversion to apathogenic organism is avoided since the vaccinia virus contains onlythe gene coding for the antigenic determinant of the disease producingorganism and not those genetic portions of the organism responsible forthe replication of the pathogen.

The present invention offers advantages even with respect to newtechnology employing genetic engineering involving the production of anantigen by a recombinant prokaryotic organism containing a plasmidexpressing a foreign antigenic protein. For instance, such technologyrequires the production of large amounts of the recombinant prokaryoticcells and subsequent purification of the antigenic protein producedthereby. In contrast, a modified vaccinia virus used for innoculationaccording to the present invention replicates within the innoculatedindividual to be immunized thereby amplifying the antigenic determinantin vivo.

A further advantage of the use of vaccinia mutants as vectors ineukaryotic cells as vaccines or for producing biological products otherthan antigens is the possibility for post-translational modifications ofproteins produced by the transcription of exogenous genes introducedinto the cell by the virus. Such post-translational modifications, forinstance glycosylation of proteins, are not likely in a prokaryoticsystem, but are possible in eukaryotic cells where additional enzymesnecessary for such modifications are available. A further advantage ofthe use of vaccinia mutants for inoculation is the possibility ofamplification of the antibody response by the incorporation, into themutant, of tandem repeats of the gene for the antigen or of additionalgenetic elements which stimulate the immune response, or by the use of astrong promoter in the modified virus. A similar advantage holds in theproduction of biological products other than antigens.

Returning to a more detailed consideration of the vaccinia genome, thecross-linked double strands of the DNA are characterized by invertedterminal repeats each approximately 8.6 megadaltons in length,representing about 10 kilobasepairs (kbp). Since the central portions ofthe DNA of all pox viruses are similar, while the terminal portions ofthe viruses differ more strongly, the responsibility of the centralportion for functions common to all the viruses, such as replication, issuggested, whereas the terminal portions appear responsible for othercharacteristics such as pathogenicity, host range, etc. If such a genomeis to be modified by the rearrangement or removal of DNA fragmentstherefrom or the introduction of exogenous DNA fragments thereinto,while producing a stable viable mutant, it is evident that the portionof the naturally-occurring DNA which is rearranged, removed, ordisrupted by the introduction of exogenous DNA thereinto must benon-essential to the viability and stability of the host, in this casethe vaccinia virus. Such non-essential portions of the genome have beenfound to be present in the WR strain of vaccinia virus, for instancewithin the region present within the L-variant but deleted from theS-variant or within the Hind III F-fragment of the genome.

The modification of vaccinia virus by the incorporation of exogenousgenetic information can be illustrated by the modification of the WRstrain of vaccinia virus in the Hind III F-fragment thereof toincorporate into that fragment a gene of herpes simplex virus type I(HSV) responsible for the production of thymidine kinase (TK). TK is anenzyme which phosphorylates the nucleoside thymidine to form thecorresponding mono-phosphorylated nucleotide which is subsequentlyincorporated into DNA.

The HSV TK gene represents DNA foreign to vaccinia virus which isconvenient to introduce into vaccinia according to the present inventionfor a number of reasons. First, the gene is relatively readily availablepresent in a herpes simplex virus DNA fragment that is produced bydigestion with Bam HI endonuclease, as reported by Colbere-Garapin etal. in Proc. Natl. Acad. Sci. USA 76, 3755-3759 (1979). Second, this HSVBam HI fragment has been introduced into plasmids and into eukaryoticsystems in the prior art, for instance as reported by Colbere-Garapin etal., loc. cit. and by Wigler et al., Cell 11, 223-232 (1977). Third,experience has shown that if HSV TK can be introduced as an exogenousgene into a eukaryotic system, and is expressed--which requiresunambiguous and faithful translation and transcription of the geneticlocus--, then other exogenous genes can similarly be introduced andexpressed.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be had by referringto the accompanying drawings, in which

FIG. 1 is a map of the aforementioned L- and S-variants of the WR strainof vaccinia determined using Hind III as a restriction enzyme andshowing the deletion of sequences in the terminal C fragment of theL-variant, which deletion is outside the terminal repeat section of thegenome. The deleted DNA sequences are unique to the L structure and,since the growth of the S- and L-variants is identical, this deletedregion must be non-essential;

FIG. 2 shows the vaccinia Hind III F-fragment in greater detailincluding two further restriction sites therein, namely Sst I and BamHI, at least the latter of which sites offers a locus at which exogenousDNA can be introduced into the vaccinia Hind III F-fragment withoutdisturbing any essential vaccinia genes;

FIGS. 3 A-C schematically show a method for the introduction of the HSVTK gene into the vaccinia Hind III F-fragment;

FIG. 4 is a restriction map of certain vaccinia mutants producedaccording to the present invention and shows in detail the position ofthe HSV TK inserts present in the Hind III F-fragment in two such virusmutants, designated herein as VP-1 and VP-2;

FIG. 5 is a table summarizing certain techniques useful in screeningpossible recombinant viruses to determine the presence or absence of theHSV TK gene therein; and

FIGS. 6 A-C are restriction maps of the left-hand terminal portion ofthe vaccinia WR genome showing the relationship of various restrictionfragments to the unique L-variant DNA sequence deleted from thecorresponding S-variant.

Referring to FIG. 1, if the L- and S-variants of the vaccinia virus aresubjected to the action of Hind III, a restriction enzyme well known inthe prior art and commercially available, the virus genomes arerespectively cleaved into 15 or 14 segments designated with the lettersA through O, with the letter A used to designate the largest fragmentand the letter O used to designate the smallest. The electrophoreticseparation of the restriction fragments is described and shown in theaforementioned publication of Panicali et al., J. Virol. 37, 1000-1010(1981). The F-fragment obtained in this manner from either the L- orS-variants has a molecular weight of 8.6 megadaltons. The position ofthe F-fragment is shown on the restriction map presented as FIG. 1accompanying the application and a restriction map of the F-fragment isshown in FIG. 2. The restriction enzyme Hind III recognizes thenucleotide sequence --AAGCTT-- and cleaves the DNA between the adjacentadenosine groups to give fragments having "sticky ends" with thesequence AGCT--. Since larger quantities of the Hind III F-fragment ofvaccinia than are readily obtainable by restriction of the vacciniagenome are required for manipulation according to the present invention,the F-fragment is inserted into a plasmid cloning vector for purposes ofamplification.

Namely, the vaccinia Hind III F-fragment produced in this manner isconveniently introduced into the plasmid pBR 322 which is cut only onceby a number of restriction enzymes, including Hind III. The pBR 322plasmid was first described by Bolivar et al. in Gene 2, 95-113 (1977)and is now commercially available within the United States from a numberof sources.

The location of the Hind III cleavage site on the pBR 322 plasmid isindicated in FIG. 3A relative to cleavage sites of Eco RI and Bam HI,which are other restriction enzymes. If the pBR 322 plasmid is cut withHind III and the resultant cleaved DNA is mixed with vaccinia Hind IIIF-fragment, and if the fragments are ligated with T₄ DNA ligase, assuggested in FIG. 3A, the F-fragment is incorporated into the plasmid toproduce the novel plasmid pDP 3 shown schematically in FIG. 3B andhaving a molecular weight of approximately 11.3 megadaltons. Thevaccinia Hind III F-fragment includes approximately 13 kilobasepairs incomparison with the 4.5 kilobasepairs found in the pBR 322 portion ofpDP 3. T₄ DNA ligase is a commercially available enzyme and theconditions for its use in the manne indicated are well known in the art.

The pDP 3 plasmid is now introduced into a microorganism such asEscherichia coli (E. coli) by transformation for purposes of replicatingthe Hind III F-fragment for recovery of larger quantities of theF-fragment. These techniques of cleaving a plasmid to produce linear DNAhaving ligatable termini and then inserting exogenous DNA havingcomplementary termini in order to produce a replicon (in this case thepBR 322 containing vaccinia Hind III F-fragment) are known in the art,as is the insertion of the replicon into a microorganism bytransformation (cf. U.S. Pat. No. 4,237,224).

Unmodified pBR 322 plasmid confers ampicillin resistance (Amp®) andtetracycline resistance (Tet®) to its host microorganism, in this caseE. coli. However, since Hind III cuts the pBR 322 plasmid in the Tet®gene, the introduction of the vaccinia Hind III F-fragment destroys theTet® gene and tetracycline resistance is lost. Hence, the E. colitransformants containing the pDP 3 plasmid can be distinguished fromuntransformed E. coli by the simultaneous presence of resistance toampicillin and susceptibility to tetracycline. It is these E. colitransformed with pDP 3 which are grown in large quantities and fromwhich large quantities of the pDP 3 are recovered.

The conditions under which plasmids can be amplified in E. coli are wellknown in the art, for example from the paper of Clewel, J. Bacteriol.110, 667-676 (1972). The techniques of isolating the amplified plasmidfrom the E. coli host are also well known in the art and are described,for instance, by Clewel et al. in Proc. Natl. Acad. Sci. USA 62,1159-1166 (1969).

In a similar fashion, the pBR 322 plasmid can be conveniently cleaved bytreatment with the restriction enzyme Bam HI and a modified plasmid canbe prepared by the insertion thereinto of a Bam HSV TK fragment, all asdiscussed in the aforementioned work of Colbere-Garapin et al., loc.cit. The modified plasmid containing the Bam HI fragment which includesthe HSV TK gene can again be introduced into E. coli by known methodsand the transformed bacteria grown for amplification of the plasmid inlarge quantities. The amplified Bam HSV TK-pBR 322 recombinant plasmidis subsequently cleaved with Bam HI to isolate the Bam HI fragmentcontaining the HSV TK gene using the same prior art techniques mentionedearlier with regard to the amplification of the Hind III F-fragment ofvaccinia.

To construct a recombinant plasmid having the Bam HI HSV TK fragmentincluded within the vaccinia Hind III F-fragment, the pDP 3 plasmid isnext subjected to a partial restriction with Bam HI such that only oneof the two Bam HI cleavage sites within the plasmid is cleaved, i.e.either that Bam HI site within the Hind III F-fragment or the Bam HIsite within the pBR 322 portion of the pDP 3 plasmid, as shown in FIG.3B. The cleaved, now-linear, DNA is then combined with purified Bam HSVTK fragment. The linear segments are combined and ligated by treatmentwith T₄ DNA ligase, again using techniques known in the art.

The combination of the Bam HSV TK fragment with the cleaved pDP 3plasmid is a random or statistical event leading to the possibleproduction of numerous species formed by various combinations of thefragments present in the mixture, all of which have identical "stickyends". Thus, one possibility is the simple rejoining of the Bam HIcleaved ends of the pDP 3 plasmid to reform the circular plasmid.Another possibility is the joinder of two or more Bam HSV TK fragmentsin either of two orientations. Further, the Bam HSV TK fragment (or amultiple thereof) may be combined with the linear DNA of a pDP 3 plasmidwhich has been cleaved at the Bam HI site within the pBR 322 portion,again in either of two orientations, or one or more Bam HSV TK fragmentsmay be combined, again in either of two orientations, with linear pDP 3DNA which has been cleaved at the Bam HI site within the vaccinia HindIII F-fragment portion of the pDP 3 plasmid.

To permit the identification and separation of these variouspossibilities, the products of ligation are inserted into a unicellularmicroorganism such as E. coli by techniques like those described earlierand known in the art. The E. coli thus treated are then grown on amedium containing ampicillin. Those bacteria which contain any plasmidare ampicillin resistant because all such plasmids contain that gene ofpBR 322 which confers ampicillin resistance. Hence, all survivingbacteria are transformants which are then screened further to determinethe presence or absence of the Bam HSV TK fragment possibly present.

To accomplish this, those bacteria containing any TK gene are identifiedby hybridization with radio-labelled TK DNA. If the TK gene is presentin the bacterium the radio-labelled TK DNA will hybridize with thatportion of the plasmid present in the bacterium. Since the hybrid isradioactive, the colonies containing TK within their plasmids can bedetermined by means of autoradiography. The bacteria containing TK canin turn be grown. Finally, then, bacteria containing plasmids having theTK incorporated within the pBR 322 portion can be identified andseparated from those having the TK fragment in the vaccinia Hind IIIF-fragment by analysis with restriction endonucleases.

More in detail, the bacteria surviving growth on nutrient agar platescontaining ampicillin are partially transferred to a nitrocellulosefilter by contact of the filter with the plate. The bacteria remainingon the plate are regrown and the bacteria which have been transferred tothe nitrocellulose filter to create a replica of the original plate arenext treated to denature their DNA. Denaturation is effected, forexample, by treatment of the transferred bacteria with sodium hydroxidefollowed by neutralization and washing. Subsequently, the now-denaturedDNA present on the nitrocellulose filter is hybridized by treatment withHSV Bam TK containing radioactive ³² P. The nitrocellulose filter sotreated is next exposed to X-ray film which darkens in those portions inwhich hybridization with the radio-labelled Bam HSV TK has taken place.The exposed darkened X-ray film is next compared with the original plateand those colonies growing on the original plate corresponding to thecolonies causing darkening of the X-ray film are identified as thosecontaining a plasmid in which Bam HSV TK is present.

Finally, to discriminate between those bacteria containing a plasmid inwhich the Bam HSV TK gene has been incorporated within the pBR 322portion of the plasmid from those wherein Bam HSV TK is present in theF-fragment of the plasmid, small cultures of the bacteria are grown andthe plasmids are isolated therefrom by a mini-lysis technique known inthe art and described in the paper of Holmes et al., Anal. Bioch. 114193-197 (1981). The plasmids are next digested with the restrictionenzyme Hind III which cleaves the circular plasmid at the two points oforiginal joinder of the F-fragment with the pBR 322 DNA chain. Themolecular weight of the digestion product is next determined byelectrophoresis on agarose gels with the distance of migration in thegels being a measur of the molecular weight.

If the Bam HSV TK fragment or a multiple thereof is found in theF-segment of the digested plasmid, the gel will show the presence of thepBR 322 fragment plus a second fragment having a molecular weightgreater than that of the F-fragment by the molecular weight of the BamHSV TK DNA segment or segments included therein. Conversely, if the BamHSV TK is present in the pBR 322, electrophoresis will show the presenceof an F-fragment of the usual molecular weight plus a further fragmentlarger than pBR 322 by the molecular weight of the Bam HSV TK fragmentor fragments present therein. Those bacteria in which modification withBam HSV TK has occurred in the pBR 322 portion of the plasmid arediscarded: the remaining bacteria have been modified in the F-fragmentportion of the plasmid therein. It is these plasmids which are used forincorporation of the Bam HSV TK fragment into vaccinia.

As mentioned earlier, the combination of the DNA fragments to regeneratea plasmid is a random event according to which a number of whichdifferent plasmid structures having Bam HSV TK in the F-fragment canresult.

To determine the orientation of the Bam HSV TK fragment within theF-fragment, as well as the number of such Bam HSV TK fragments possiblypresent, the plasmids are recovered from each of those bacterialcolonies which are known to have an Bam HSV TK fragment present in theF-fragment of the plasmid. The mini-lysis technique mentioned earlierherein is used for this purpose. The plasmids are then again subjectedto restriction analysis, this time using the commercially availablerestriction enzyme Sst I. Since each Bam HSV TK fragment has an Sst Irestriction site therein, and since the F-fragment of vaccinia similarlyhas a single Sst I restriction site therein (cf. the representation ofthese fragments in FIGS. 3A and 3B respectively), different numbers offragments of differing molecular weights can be detected byelectrophoresis on agarose gels, the number and molecular weight of thesegments being dependent on the orientation of the Bam HSV TK fragmentwithin the F-fragment and the number of such Bam TK fragments present.Orientation of the Bam TK fragment within the F-fragment can be detectedbecause of the asymmetry of the Bam HSV TK fragment with respect to theSst I site therein (cf. FIG. 3B).

For instance, in the particular experiments under discussion, sixbacterial colonies each having one or more Bam HSV TK fragments presentin the F-fragment of the plasmid were found among the E. colitransformants. After restriction analysis of the plasmids in thesebacteria along the lines discussed above, two of the recombinantplasmids were chosen for further study because the direction oforientation of the Bam HSV TK fragment within the F-fragment was inopposite directions.

At this point, the reader is reminded that the introduction of the HSVTK gene into the F-fragment of vaccinia, as discussed in detail above,is merely exemplary of one of many possible means of modifying thevaccinia genome to produce desirable vaccinia mutants. Thus, theintroduction of the same exogenous gene into another portion of thevaccinia genome, or the introduction of different genetic material intothe vaccinia F-fragment or into some other fragment, all may requiremodification of the exemplary scheme, discussed above, for theidentification of recombinant organisms.

For instance, digestion of the vaccinia L-variant with Ava I yields afragment, H, entirely with the region deleted from the S-variant (cf.FIG. 6 A and the discussion thereof infra). This H-fragment contains BamHI sites permitting the introduction thereinto of the HSV TK gene. Thesame scheme for identifying F-fragment-HSV TK recombinants can be usedfor identifying such H-fragment recombinants also.

Indeed, schemes for the construction and identification ofF-fragment-HSV TK recombinants, alternative to that disclosed in detailabove by way of illustration, do exist. For instance, the Bam HI site inpBR 322 can be removed by cleavage of the plasmid with Bam HI andtreatment with DNA polymerase I to "fill in" the "sticky ends". Thisproduct is then cut with Hind III and the linear fragment is treatedwith alkaline phosphatase to prevent recircularization of the plasmidupon ligation. However foreign DNA, and particularly the vaccinia HindIII F-fragment, can be ligated to the treated pBR 322 and the resultingplasmid will recircularize. Now, treatment with Bam HI effects cleavageof the plasmid only within the vaccinia F-fragment portion thereof.Subsequent treatment of the cleavage product with alkaline phosphatasean ligation with the Bam HI HSV TK fragment will produce recombinantswith high efficiency s that the recombinants can be screened byrestriction endonuclease cleavage and gel electrophoresis. Thistechnique eliminates the time-consuming steps of discriminating betweenrecombinants having HSV TK in the pBR 322 portion or in the F-fragmentand colony hybridization.

Returning now to further discussion of the plasmids produced in theexemplary mutation of vaccinia by the introduction of HSV TK into thevaccinia F-fragment the two recombinant plasmids chosen for furtherstudy are shown in FIG. 3C, where they are identified as a first novelplasmid, pDP 132, incorporating one Bam HSV TK fragment within thevaccinia Hind III F-portion, and a second novel plasmid, pDP 137, inwhich two Bam HSV TK fragments joined "head to tail" have beenincorporated. The single fragment of Bam HSV TK has been incorporatedwithin pDP 132 in the opposite sense in which two Bam TK fragments havebeen included in tande in pDP 137. Namely, the region of the TK genewithin the Bam HI fragment which codes for the 5'-end of mRNA producedby the gene is located between the Sst I cleavage site and the nearer ofthe two Bam HI sites thereto (again cf. FIG. 3B). The direction oftranscription of the HSV TK gene on the Bam TK fragment proceeds fromthe 5'-end to the 3'-end and will be in a clockwise direction in pDP 132as shown in FIG. 3C. [cf. Smiley et al., Virology 102, 83-93 (1980)].Conversely, since the Bam TK fragments included in tandem in pDP 137have been incorporated in the reverse sense, transcription of the HSV TKgenes contained therein will be in the opposite direction, namely in acounter-clockwise direction. The direction of inclusion of the Bam HSVTK fragment within the vaccinia Hind III F-fragment may be of importancein case promotion of transcription of the HSV TK gene is initiated by apromoter site within the F-fragment itself. However, HSV promoter sitesdo exist within the Bam HSV TK fragment itself, so that transcription ofthe HSV TK gene may occur no matter in which direction the Bam HSV TKfragment and HSV TK gene have been incorporated within the vaccinia HindIII F-fragment.

Those E. coli transformants containing pDP 132 or pDP 137 are next grownto produce large amounts of the plasmids for further processing. When asufficient amount of the plasmid DNA has been isolated, restriction withHind III yields a modified vaccinia Hind III F-fragment having the HSVTK gene therein. This modified Hind III F-fragment is now introducedinto vaccinia virus by novel methods, described below in greater detail,in order to produce an infectious entity.

To review the prior art, at present the vector principally used forintroducing exogenous DNA into eukaryotic cells is SV40. The DNA of SV40is circular and can be treated much like a plasmid. That is, thecircular DNA is cleaved with a restriction enzyme, combined withexogenous DNA, and ligated. The modified DNA can be introduced intoeukaryotic cells, for instance animal cells, by standard techniques [cf.Hamer et al., Nature 281, 35-40 (1979)]. The DNA is infectious and willreplicate in the nucleus of the cell producing viable mutated viruses.In contrast, vaccinia replicates within the cytoplasm of a eukaryoticcell. The purified DNA of this virus is not infectious and cannot beused per se to produce vaccinia mutants in a cell in the same manner asSV40. Rather novel techniques involving the mutation of wild typevaccinia with foreign DNA in vivo within a cell must be employed.

An unpublished paper of the applicants together with Eileen Nakano,reports a demonstration of marker rescue in vaccinia virus. According tothese experiments, that portion of the L-variant DNA which is normallyabsent from the S-variant can be reintroduced into the S-variant("rescued") under appropriate conditions. Namely, eukaryotic cells artreated with live infectious S-variant vaccinia virus together withnon-infectious restriction fragments of the DNA of the L-variant,representing DNA "foreign" to the S-variant, of a particular structure.Namely, that portion of the L-variant DNA which is to be rescued must bepresent within a DNA chain having portions co-linear with the DNA chainof the S-fragment into which it is to be introduced. That is, the"foreign" DNA to be introduced into the S-variant has at both ends ofthe DNA chain, a region of DNA which is homologous with correspondingsequences in the S-variant. These homologous sequences can be viewed as"arms" attached to the region of L-variant DNA which is to be rescued bythe S-variant.

The mechanism of this recombination is complex and has not yet beenaccomplished in vitro. Apparently, the recombination of the L-DNA intothe S-variant involves homologous base pairing in segments surroundingthe area deleted from the S-variant. Most likely, cross-overs from onestrand of DNA to another result in an in vivo recombination of the DNAto rescue the deleted portion.

This technique of in vivo recombination can be used to introduce foreignDNA other than vaccinia DNA into either the S- or the L-variant ofvaccinia. Thus, the modified Hind III F-fragment incorporating the BamHSV TK fragment therein as DNA "foreign" to vaccinia can be introducedinto vaccinia by treating eukaryotic cells with the modified F-fragmenttogether with infectious L- and/or infectious S-variants of vacciniavirus. In this instance, the portions of the F-fragment flanking the BamHSV TK fragment function as the "arms" mentioned earlier comprising DNAhomologous with DNA present in the L- or S-variant into which themodified F-fragment is to be introduced. Again, by in vivo processeswithin the cell, the mechanisms of which are not known in detail, theHSV TK-modified F-fragment is incorporated into the vaccinia variants inthe cell and is then capable of replication and expression undervaccinia control.

This in vivo recombination technique is broadly applicable to theintroduction of still other "foreign" DNA into vaccinia, providing apathway by which the genome of vaccinia can be modified to incorporate awide variety of foreign genetic material thereinto, whether such foreignDNA be derived from vaccinia itself, be synthetic, or be derived fromorganisms other than vaccinia.

A wide variety of cells can be used as the host cells in which the invivo recombination described above takes place. The recombination,however, occurs with differing efficiency depending on the cellemployed. Of the cells investigated to date, baby Syrian hamster kidneycells [BHK-21 (Clone 13) (ATCC No. CCL10)] have proved most efficientfor the recombination procedure. However, other cells including CV-1(ATCC No. CCL70), a green monkey kidney cell line, and human (line 143)TK-cells, a 5'-BUdR resistant mutant derived from human cell lineR970-5, have also been infected in this manner to generate vacciniamutants.

These cells are suitably treated with vaccinia an the foreign DNA to beincorporated into the vaccinia while, for convenience, the cells are inthe form of a monolayer. For purposes of in vivo recombination, thecells may be infected with vaccinia followed by treatment with theforeign DNA to be incorporated thereinto, or may first be contacted withthe foreign DNA followed by infection with vaccinia. As a thirdalternative, the vaccinia and foreign DNA may be simultaneously presentat the time the cells are treated.

The viruses are suitably contacted with the cell monolayer while presentin a conventional liquid medium, such as phosphate buffered saline,Hepes buffered saline, Eagle's Special medium (with or without serumaddition), etc. which is compatible with these cells and the viruses.

The foreign DNA is conveniently used to treat these cells while in theform of a calcium phosphate precipitate. Such techniques for introducingDNA into cells have been described in the prior art by Graham et al.,Virology 52, 456-467 (1973). Modifications of the technique have beendiscussed by Stow et al., J. Gen. Virol. 33, 447-458 (1976) and Wigleret al., Proc. Natl. Acad. Sci. USA 76, 1373-1376 (1979). The treatmentstaught in these papers conveniently proceed at room temperature buttemperature conditions can be varied within limits preserving cellviability, as can the time for which the cells are treated with thevirus and/or foreign DNA precipitate, with various efficiencies of thein vivo recombination. The concentration of the infecting vaccinia virusand the amount of foreign DNA precipitate employed will also affect therate or degree of recombination. Other factors such as atmosphere andthe like are all chosen with a view to preserving cell viability.Otherwise, as long as the three necessary components (cell, virus, andDNA) are present, in vivo recombination will proceed at least to someextent. Optimization of the conditions in a particular case is wellwithin the capabilities of one skilled in the microbiological arts.

Following this recombination step, those vaccinia viruses which havebeen mutated by in vivo recombination must be identified and separatedfrom unmodified vaccinia virus.

Vaccinia viruses mutated by in vivo recombination of foreign DNAthereinto can be separated from unmodified vaccinia virus by at leasttwo methods which are independent of the nature of the foreign DNA orthe ability of the mutant to express any gene which may be present inthe foreign DNA. Thus, first, the foreign DNA in the mutant genome canbe detected by restriction analysis of the genome to detect the presenceof a extra piece of DNA in the mutated organism. In this methodindividual viruses isolated from purified plaques are grown and the DNAis extracted therefrom and subjected to restriction analysis usingappropriate restriction enzymes. Again, by detecting the number andmolecular weight of the fragments determined, the structure of thegenome prior to restriction can be deduced. However, because of thenecessity of growing purified plaques, the number of analyses which mustbe made, and the possibility that none of the plaques grown and analyzedwill contain a mutant, this technique is laborious, time consuming anduncertain.

Further, the presence of foreign DNA in vaccinia virus can be determinedusing a modification of the technique taught by Villarreal et al. inScience 196, 183-185 (1977). Infectious virus is transferred from viralplaques present on an infected cell monolayer to a nitrocellulosefilter. Conveniently, a mirror-image replica of the transferred viruspresent on the nitrocellulose filters is made by contacting a secondsuch filter with that side of the first nitrocellulose filter to whichthe viruses have been transferred. A portion of the viruses present onthe first filter is transferred to the second filter. One or the otherof the filters, generally the first filter, is now used forhybridization. The remaining filter is reserved for recovery ofrecombinant virus therefrom once the locus of the recombinant virus hasbeen detected using the hybridization technique practiced on thecompanion, mirror-image filter.

For purposes of hybridization, the viruses present on the nitrocellulosefilter are denatured with sodium hydroxide in a manner known per se. Thedenatured genetic material is now hybridized with a radio-labelledcounterpart of the gene whose presence is sought to be determined. Forexample, to detect the possible presence of vaccinia mutants containingthe Bam HSV TK fragment, the corresponding radio-labelled Bam HSV TKfragment containing ³² P is employed, much in the same manner asdiscussed earlier herein with respect to the detection of plasmidsmodified by the presence of this fragment. Non-hybridized DNA is washedfrom the nitrocellulose filter and the remaining hybridized DNA, whichis radioactive, is located by autoradiography, i.e. by contacting thefilter with X-ray film. Once the mutated viruses are identified, thecorresponding virus plaques present on the second filter, containing amirror image of the viruses transferred to the first filter, are locatedand grown for purposes of replicating the mutated viruses.

The two methods described above involve a analysis of the genotype ofthe organism involved and, as mentioned earlier, can be used whether ornot any gene present within the foreign DNA incorporated into thevaccinia virus is expressed. However, if the foreign DNA is expressed,then phenotypic analysis can be employed for the detection of mutants.For example, if the gene is expressed by the production of a protein towhich an antibody exists, the mutants can be detected by a methodemploying the formation of antigen-antibody complexes. See Bieberfeld etal. J. Immunol. Methods 6, 249-259 (1975). That is, plaques of theviruses including the suspected mutants are treated with the antibody tothe protein which is produced by the mutan vaccinia genotype. Excessantibody is washed from the plaques, which are then treated with proteinA labelled with ¹²⁵ I. Protein A has the ability of binding to the heavychains of antibodies, and hence will specifically label theantigen-antibody complexes remaining on the cell monolayer. After excessradioactive protein A is removed, the monolayers are again picked up byplaque lifts onto nitrocellulose filters and are subjected toautoradiography to detect the presence of the radio-labelled immunecomplexes. In this way, the mutated vaccinia viruses producing theantigenic protein can be identified.

In the specific instance in which the foreign DNA includes the HSV TKgene, once it is known that the mutated vaccinia virus expresses the HSVTK gene therein, a much simpler and elegant means for detecting thepresence of the gene exists. Indeed, the ease of discrimination betweenvaccinia mutants containing the HSV TK gene and unmodified vaccinia freeof this gene provides a powerful tool for discriminating betweenvaccinia virus mutants containing other exogenous genes either presentalone in the vaccinia genome or present therein in combination with theHSV TK gene. These methods are described more in detail later herein.

Since eukaryotic cells have their own TK gene and vaccinia virussimilarly has its own TK gene (utilized, as noted above, for theincorporation of thymidine into DNA), the presence and expression ofthese genes must be in some way distinguished from the presence andexpression of the HSV TK gene in vaccinia mutants of the type underdiscussion. To do this, use is made of the fact that the HSV TK genewill phosphorlyate halogenated deoxycytidine, specificallyiododeoxycytidine (IDC), a nucleoside, but neither the TK gene ofvaccinia nor the TK gene of cells will effect such a phosphorylation.When IDC is incorporated into the DNA of a cell it becomes insoluble.Non-incorporated IDC, on the other hand, is readily washed out from cellcultures with an aqueous medium such as physiologic buffer. Use is madeof these facts as follows to detect the expression of the HSV TK gene invaccinia mutants.

Namely, cell monolayers are infected with mutated virus under conditionspromoting plaque formation, i.e. those promoting cell growth and virusreplication. When the cells are infected, they are then treated withcommercially available radio-labelled IDC (IDC*), labelling being easilyeffected with ¹²⁵ I. If the cells are infected with a virus containingthe HSV TK gene, and if the HSV TK gene present therein is expressed,the cell will incorporate IDC* into its DNA. If the cell monolayers arenow washed with a physiologic buffer, non-incorporated IDC* will washout. If the cell monolayers are next transferred to a nitrocellulosefilter and exposed to X-ray film, darkening of the film indicates thepresence of IDC* in the plaques an demonstrates the expression of theHSV TK gene by the vaccinia mutants.

Using the aforementioned genotypic and phenotypic analyses theapplicants have identified two vaccinia mutants denominated VP-1 andVP-2. VP-1 (ATCC No. VR 2032) is a recombinant vaccinia virus derivedfrom vaccinia S-variant modified by in vivo recombination with theplasmid pDP 132. VP-2 (ATCC No. VR 2030) is an S-variant vaccinia virusmodified by recombination with pD 137.

FIG. 4A is a Hind III restriction map of the vaccinia genome showing thesite of the HSV TK gene insertion. FIGS. 4B and 4C magnify the Hind IIIF-fragment respectively contained in VP-1 and VP-2 to show theorientation of the Bam HI HSV TK fragment therein. Attention is calledto the fact that the in vivo recombination of pDP 137 with the S-variant(i.e. VP-2) effects deletion of one of the Bam HI HSV TK fragmentspresent in tandem in the starting plasmid.

As mentioned earlier, the fact that the HSV TK gene is expressed can beused for a rapid and easy detection and identification of mutants whichcontain or are free of HSV TK gene or of a foreign gene present alone orin combination with the HSV gene. The test and its bases are describedimmediately below.

The applicants have isolated in biologically pure form, a vacciniamutant, an S-variant in particular, which is free of anynaturally-occurring functional TK gene, denominated VTK⁻ 79 (ATTC No. VR2031). Normally, the S- and L-variants discussed earlier herein have aTK gene in the Hind III fragment J thereof. If this mutant, free ofvaccinia TK gene activity, is used for the production of further mutatedorganisms containing the HSV TK gene, incorporated into the vacciniamutant by the techniques described earlier herein, the HSV TK genepresent in such resultant mutants will be the only functional TK genepresent in the virus. The presence or absence of such an HSV TK gene canbe immediately detected by growing cells infected with the viruses onone of several selective media.

Namely, one such selective medium contains bromodeoxyuridine (BUdR), anucleoside analogous to thymidine, but highly mutagenic and poisonous toorganisms such as a cell or virus when present in DNA contained therein.Such a medium is known from Kit et al., Exp. Cell Res. 31, 297-312(1963). Other selective media are the hypoxanthine/aminopterin/thymidine(HAT) medium of Littlefield, Proc. Natl. Acad. Sci. USA 50, 568-573(1963) and variants thereof such as MTAGG, described by Davis et al., J.Virol. 13, 140-145 (1974) or the further variant of MTAGG described byCampione-Piccardo et al. in J. Virol. 31, 281-287 (1979). All thesemedia selectively discriminate between organisms containing andexpressing a TK gene and those which do not contain or express any TKgene. The selectivity of the media is based on the following phenomena.

There are two metabolic pathways for the phosphorylation of thymidine.The primary metabolic pathway does not rely upon thymidine kinase and,while it synthesizes phosphorylated thymidine by intermediatemechanisms, it will not phosphorylate BUdR or directly phosphorylatethymidine The secondary metabolic pathway does involve the activity ofthymidine kinase and will result in the phosphorylation of boththymidine and its analog, BUdR. Since BUdR is a poisonous highlymutagenic substance, the presence of TK, such as the HSV TK underdiscussion, in an organism will result in the phosphorylation of BUdRand its incorporation into the DNA of the growing organism, resulting inits death. On the other hand, if the TK gene is absent or not expressed,and the primary metabolic pathway which then is followed results in thesynthesis of phosphorylated thymidine but not in the phosphyorylation ofBUdR, the metabolizing organism will survive in the presence of BUdRsince this substance is not incorporated into its DNA.

The growth behaviors discussed above are summarized in FIG. 5 of theaccompanying drawings tabulating the growth behavior or organismsexpressing TK (TK⁺) and organisms free of or not expressing the TK gene(TK⁻) on a normal medium, on a selective medium such as HAT which blocksthe primary metabolic pathway not using TK, and on a medium containingBUdR. TK⁺ and TK⁻ organisms will both grow on a normal growth medium byemploying the primary metabolic pathway not requiring TK. On a selectivemedium such as HAT which blocks the primary metabolic pathway notrelying on TK, the TK⁺ organism will nevertheless grow because theenzyme accomplishes the phosphorylation necessary for incorporation ofthymidine into DNA. On the other hand, the TK⁻ organisms will notsurvive. In contrast, if the organisms are grown on a medium containingBUdR, the TK⁺ variants will die since TK phosphorylates BUdR and thispoisonous material is incorporated in the DNA. In contrast, since BUdRis not phosphorylated by the primary metabolic pathway, the TK⁻ variantwill grow since BUdR is not incorporated into the DNA.

Thus, if a vaccinia virus free of vaccinia TK, such as VTK⁻ 79, is usedas the vaccinia virus into which the HSV TK gene is inserted by thetechniques of the present invention, the presence and expression, or theabsence, of the HSV TK gene therein can be readily determined by simplygrowing the recombinants on a selective medium such as HAT. Thoseviruses which are mutated will survive since they use the HSV TK tosynthesize DNA.

The applicants have indeed prepared several mutants of vaccinia virusfree of vaccinia TK. These have been denominated VP-3 (ATCC No. VR2036), a recombinant of VTK⁻ 79 and pDP 132, and VP-4 (ATCC No. VR2033), a recombinant of VTK⁻ 79 and pDP 137. The latter expresses theHSV gene and can readily be identified using the selective mediamentioned above.

Two additional recombinant viruses, denominated VP-5 (ATCC No. VR 2028),and VP-6 (ATCC No. VR 2029), are respectively recombinants of pDP 132and pDP 137 with VTK⁻ 11 (ATCC No. VR 2027), a known L-variant ofvaccinia which does not express the vaccinia TK gene. Thus, DNA can beintroduced in excess of the maximum vaccinia genome length.

The techniques of the present invention can be used to introduce the HSVTK gene into various portions of the vaccinia genome for purposes ofidentifying non-essential portions of the genome. That is, if the HSV TKgene can be inserted into the vaccinia genome, as it is in the Hind IIIF-fragment thereof, the region of the genome into which it has beenintroduced is evidently non-essential. Each non-essential site withinthe genome is a likely candidate for the insertion of exogenous genes sothat the methods of the present invention are useful in mapping suchnon-essential sites in the vaccinia genome.

Further if the HSV TK gene is coupled with another exogenous gene andthe resultant combined DNA material is put into a vaccinia virus free ofvaccinia TK gene, such as VTK⁻ 79, recombinants which are formed andwhich contain the foreign gene will express the HSV TK gene and can bereadily separated from the TK⁻ variants by the screening techniquedescribed immediately above

A further embodiment of the invention involves the preparation of avaccinia Hind III F-fragment containing an exogenous gene therein andthe treatment of cells with the fragment together with a vaccinia mutantnot expressing the vaccinia TK gene but having the HSV TK geneincorporated therein by in vivo recombination according to thetechniques of the present invention. As with the marker rescue mentionedearlier herein and the in vivo techniques employed to incorporate theTK-modified Hind III F-fragment into vaccinia, cross-over andrecombination can occur to produce a further mutant in which the HSV Tmodified F-fragment is replaced by an F-fragment containing anotherexogenous gene. The resulting vaccinia mutant, in which the HSV TKF-fragment has been replaced by a F-fragment containing the exogenousgene, will be totally free of TK, whereas the non-mutated parent viruspredominantly present will still be HSV TK⁺. Similarly a foreign genemay be inserted into the HSV TK gene present in such a vaccinia mutant,disrupting the integrity of the gene rendering the recombinant organismTK⁻ in comparison with the non-mutated TK⁺ parent. In both instances, animmediate discrimination can be made between those vaccinia mutantscontaining the foreign gene and those which are free of any TK by growthon BUdR and/or a special medium such as HAT.

A better understanding of the present invention and of its manyadvantages will be had by referring to the following specific Examples,given by way of illustration. The percentages given are percent byweight unless otherwise indicated.

EXAMPLE I Isolation of Vaccinia Hind III Fragments from Agarose Gels

Restriction endonuclease Hind III was purchased from Boehringer MannheimCorp. Preparative digestions of DNA were performed in 0.6 ml of Hind IIIbuffer containing 10 millimolar (mM) Tris-HCl (pH 7.6), 50 mM NaCl, 10mM MgCl₂, 14 mM dithiothreitol (DTT), and 10 micrograms (μg)/ml ofbovine serum albumin (BSA) in which are present 10-20 μg of vaccinia DNAand 20-40 units of Hind III (1 unit is the amount of enzyme sufficientto cleave 1 ug of lambda-DNA completely in 30 minutes.)

Vaccinia DNA was extracted and purified from virions as follows.Purified virions were lysed at a concentration having an optical densityper ml of 50 measured at 260 nanometers (A₂₆₀) in 10 mM Tris-HCl (pH7.8), 50 mM beta-mercaptoethanol, 100 mM NaCl, 10 mM Na₃ EDTA, 1%Sarkosyl NL-97, and 26% sucrose. Proteinase K was added to 100 μg/ml andthe lysate incubated at 37° C. overnight. DNA was extracted by theaddition of an equal volume of phenol-chloroform (1:1). The organicphase was removed and the aqueous phase reextracted until the interfacewas clear. Two additional extractions with chloroform were performed andthe aqueous phase was then dialyzed extensively against 10 mM Tris-HCl(pH 7.4) containing 0.1 mM Na EDTA at 4° C. DNA was concentrated toapproximately 100 μg/ml with Ficoll (a synthetic high copolymer ofsucrose and epichlorohydrin).

Digestion of the DNA was for 4 hours at 37° C. The reactions wereterminated by heating to 65° C. for 10 minutes followed by addition ofan aqueous stop solution containing 2.5% of agarose, 40% of glycerol, 5%of sodium dodecyl sulfate (SDS), and 0.25% of bromophenol blue (BPB).Samples were layered at 65° C. onto agarose gel and allowed to hardenprior to electrophoresis.

Electrophoresis was carried out in 0.8% agarose gels (0.3×14.5×30 cm) inelectrophoresis buffer containing 36 mM Tris-HCl (pH 7.8), 30 mM NaH₂PO₄, and 1 mM EDTA. Electrophoresis was at 4° C. for 42 hours at 50volts. The gels were stained with ethidium bromide (1 μg/ml inelectrophoresis buffer). The restriction fragments were visualized withultraviolet (UV) light and individual fragments were cut from the gel.

Fragments were separated from the agarose gel according to the procedureof Vogelstein et al., Proc. Natl. Acad. Sci. USA 76, 615-619 (1979)using glass powder as follows. The agarose gel containing a DNA fragmentwas dissolved in 2.0 ml of a saturated aqueous solution of NaI. 10 mg ofglass powder were added per μg of DNA calculated to be present. Thesolution was rotated at 25° C. overnight to bind the DNA to the glasspowder. The DNA-glass powder was collected by centrifugation at 2000 rpmfor 5 minutes. The DNA-glass was then washed with 5 ml of 70% NaI. TheDNA-glass was again collected by centrifugation and washed in a mixtureof 50% buffer [20 mM Tris-HCl (pH 7.2), 200 mM NaCl, 2 mM EDTA] and 50%ethanol. The DNA-glass was collected again by centrifugation and wasgently suspended in 0.5 ml of 20 mM Tris-HCl (pH 7.2), 200 mM NaCl, and2 mM EDTA. The DNA was then eluted from the glass powder at 37° C. byincubation for 30 minutes. The glass was then removed by centrifugationat 10,000 rpm for 15 minutes. DNA was recovered from the supernatant byethanol precipitation and dissolved in 10 mM Tris-HCl (pH 7.2)containing 1 mM EDTA.

The F-fragment isolated in this way was used in the following Examples.

EXAMPLE II Insertion of the Vaccinia Hind III-F Fragment Into the HindIII Site of pB 322 (Construction of pDP 3 [pBR 322-Vaccinia Hind IIIF-Recombinant Plasmid])

Vaccinia Hind III-F fragment was isolated from preparative agarose gelsas described in Example I. This fragment was inserted into the Hind IIIsite of pBR 322 [Bolivar et al., Gene, 2, 95-113 (1977)] as follows.

Approximately 200 nanograms (ng) of pBR 322 were cleaved with Hind IIIin 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 10 mM MgCl₂, and 14 mM DTT [HindIII buffer] using 1 unit of enzyme for 1 hour at 37° C. The reaction wasstopped by heating to 65° C. for 10 minutes 500 ng of isolated Hind IIIvaccinia F-fragment were added and the DNAs co-precipitated with 2volumes of ethanol at -70° C. for 30 minutes. The DNA was then washedwith 70% aqueous ethanol, dried, and resuspended in ligation bufferconsisting of 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 10 mM DTT, and 1 mMadenosine triphosphate (ATP). Approximately 100 units of T₄ DNA ligase(New England Biolabs) were then added and the mixture was incubated at10° C. overnight. The ligase-treated DNA was then used to transform E.coli HB101 [Boyer et al., J. Mol. Biol. 41, 459-472 (1969)].

EXAMPLE III Transformation of E. coli and Selection for RecombinantPlasmids

Competent cells were prepared and transformed with plasmids according tothe procedure described by Dagert e al., Gene 6, 23-28 (1979). E. coliHB101 cells were made competent by inoculating 50 ml of LB broth (1% ofbacto-tryptone, 0.5% of bacto-yeast extract, and 0.5% of NaClsupplemented with 0.2% of glucose) with 0.3 ml of an overnight cultureof the cells and allowing them to grow at 37° C. until the culture hadan optical density (absorbence), at 650 nanometers (A₆₅₀), of 0.2, asmeasured with a spectrophotometer. The cells were then chilled on icefor 10 minutes, pelleted by centrifugation, resuspended in 20 ml of cold0.1 molar (M) CaCl₂, and incubated on ice for 20 minutes. The cells werethen pelleted and resuspended in 0.5 ml of cold 0.1 M CaCl₂ and allowedto remain at 4° C. for 24 hours. The cells were transformed by addingligated DNA (0.2-0.5 mg in 0.01-0.02 ml of ligation buffer) to competentcells (0.1 ml). The cells were then incubated on ice for 10 minutes andat 37° C. for 5 minutes. 2.0 ml of LB broth were then added to the cellsand incubated at 37° C. for 1 hour with shaking. Aliquots of 10microliters (μl) or 100 μl were then spread on LB agar plates containingampicillin (Amp) at a concentration of 100 ug/ml

The transformed bacteria were then screened for recombinant plasmids bytransferring ampicillin resistant (Amp^(R)) colonies to LB agarcontaining tetracycline (Tet) at 15 ug/ml. Those colonies which wereboth Amp^(R) and tetracycline sensitive (Tet^(S)) (approximately 1%)were screened for intact vaccinia Hind III-F fragment inserted into pBR322 according to the procedure of Holmes et al., Anal. Bioch. 114,193-197 (1981). 2.0 ml cultures of transformed E. coli were grownovernight at 37° C. The bacteria were pelleted by centrifugation andresuspended in 105 ul of a solution of 8% sucrose, 5% Triton X-100, 50mM EDTA, and 50 mM Tris-HCl (pH 8.0), followed by the addition of 7.5 μlof a freshly prepared solution of lysozyme (Worthington Biochemicals)[10 mg/ml in 50 m Tris-HCl(pH 8.0)]. The lysates were placed in aboiling water bath for 1 minute and then centrifuged at 10,000 rpm for15 minutes. The supernatant was removed and plasmid DNA precipitatedwith an equal volume of isopropanol. The plasmids were then resuspendedin 40 μl of Hind III buffer and digested with 1 unit of Hind III for 2hours. The resulting digests were then analyzed on a 1.0% analyticalagarose gel for the appropriate Hind III-F fragment. One suchrecombinant plasmid containing an intact Hind III-F fragment,denominated pDP 3, was used for further modification. (See FIG. 3B).

EXAMPLE IV Preparative Isolation of pDP 3

Large scale isolation and purification of plasmid DNA was performed by amodification of the procedure of Clewel et al., Proc. Natl. Acad. Sci.USA 62, 1159-1166 (1969). 500 ml of LB broth were inoculated with 1.0 mlof an overnight culture of E. coli HB 101 containing pDP 3. At anoptical density (A₆₀₀) of approximately 0.6, chloramphenicol was added(100 μg/ml) to amplify the production of plasmids [Clewel, J. Bacteriol.110, 667-676 (1972)]. The bacteria were incubated at 37° C. for anadditional 12-16 hours at which time they were collected bycentrifugation at 5000 rpm for 5 minutes, washed once in 100 ml of TENbuffer [0.1 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA], collected bycentrifugation and resuspended in 14 ml of a 25% solution of sucrose in0.05 M Tris-HCl (pH 8.0). 4.0 ml of lysozyme solution [5 mg/ml in 0.25 MTris-HCl (pH 8.0)] were added and the mixture was incubated at roomtemperature for 30 minutes followed by the addition of 4.0 ml of 0.25 MEDTA (pH 8.0). The mixture was then put on ice for 10 minutes. 2.0 ml ofpancreatic RNase A (Sigma Chemical Co. ) [1 mg/ml in 0.25 M Tris-HCl (pH8.0)] were added to this mixture, which is then incubated at roomtemperature for 1 minute. The cells were then lysed by adding 26 ml of alytic Triton solution [1% Triton X-100, 0.05 M EDTA, 0.05 M Tris-HCl (pH8.0)]. The mixture was incubated at room temperature for 30-60 minutes.The lysate was cleared by centrifugation at 17,000 rpm for 30 minutes at4° C. The supernatant wa then removed and plasmid DNA separated fromchromosomal DNA on dye-bouyant CsCl gradients.

For this purpose, CsCl-ethidium bromide gradients were prepared bydissolving 22 g of CsCl in 23.7 ml of cleared lysate. 1.125 ml ofaqueous ethidium bromide (10 mg/ml) were added to the solution. Themixture was then centrifuged in polyallomer tubes in a Beckman 60 Tirotor at 44,000 rpm for 48-72 hours. The resulting bands of DNA in thegradients were visualized with ultraviolet light and the lower band(covalently closed plasmid DNA) was removed by puncturing the tube withan 18 gauge needle attached to a syringe. Ethidium bromide was removedfrom the plasmid by repeated extraction with 2 volumes ofchloroform-isoamyl alcohol (24:1). Plasmids were then dialyzedextensively against 10 mM Tris-HCl (pH 7.4) containing 0.1 mM EDTA toremove CsCl. The plasmid DNA was then concentrated by ethanolprecipitation.

EXAMPLE V Construction of pBR 322/Vaccinia/Herpes Virus TK RecombinantPlasmids

FIGS. 3B and 3C summarize the steps involved in the construction of therecombinant plasmids used for inserting the Bam HSV TK fragment into S-or L-variant vaccinia. Approximately 15 ug of covalently closed pDP 3were cleaved by partial digestion with Bam HI (Bethesda ResearchLaboratories) by incubation in Bam HI buffer, consisting of 20 mMTris-HCl (pH 8.0), 7 mM MgCl₂, 100 mM NaCl, and 2 mMbeta-mercaptoethanol, using 7 units of Bam HI for 10 minutes at 37° C.Since pBR 322 and vaccinia Hind III F each contain a Bam HI site,partial cleavage results in a mixture of linear plasmids cut either atthe pBR 322 or vaccinia Bam HI site. These mixed linear plasmids werethen separated from the fragments of pDP 3 cut at both the pBR 322 andvaccinia Bam HI sites by electrophoresis on agaros gels and the singlycut linear plasmids were isolated using glass powder as described inExample I.

A recombinant pBR 322 containing the 2.3 megadalton (md) HSV Bam HIfragment which codes for HSV TK, as described by Colbere-Garapin et al.,Proc. Natl. Acad. Sci. USA 76, 3755-3759 (1979), was digested tocompletion with Bam HI and the 2.3 md Bam TK fragment was isolated froman agarose gel as described above.

pDP 3 Bam TK recombinant plasmids were constructed by ligatingapproximately 1 μg of Bam HI linearized pDP 3 to approximately 0.2 μg ofisolated Bam TK fragment in 20 μl of ligation buffer containing 100units of T4 DNA ligase at 10° C. overnight. This ligation mixture wasthen used to transform competent E. coli HB 101 cells as described inExample III.

EXAMPLE VI Screening of Transformed Cells for Identification of ThoseContaining Recombinant Plasmids Having HSV TK Inserts

Transformed cells containing recombinant plasmids were screened for HSVTK insertions by colony hybridization essentially as described byHanahan et al., Gene 10, 63-67 (1980).

A first set of nitrocellulose filters (Schleiche and Schull BA85) wereplaced on Petri dishes filled with LB agar containing 100 μg/ml ofampicillin. Transformed cells were spread on the filters and the disheswere incubated at 30° C. overnight or until the colonies were justvisible. A replica nitrocellulose filter of each of the first set offilters was made by placing a sterile nitrocellulose filter on top ofeach of the above-mentioned original filters and pressing the twofilters together firmly. Each pair of filters was then notched (keyed)with a sterile scalpel blade, separated, and each filter was transferredto a fresh LB agar plate containing ampicillin at 100 μg/ml for 4-6hours. The first set of filters (original filters) were then placed onLB agar plates containing 200 μg/ml of chloramphenicol to amplifyplasmid production. The replica filters were stored at 4° C.

After 24 hours on chloramphenicol, the original nitrocellulose filterswere prepared for hybridization as follows. Each nitrocellulose filterwas placed on a sheet of Whatman filter paper saturated with 0.5 N NaOHfor 5 minutes, blotted on dry filter paper for 3 minutes, and placedback on the NaOH saturated filter paper for 5 minutes to lyse thebacteria thereon and to denature their DNA. This sequence was thenrepeated using Whatman filter paper sheets saturated with 1.0 M Tris-HCl(pH 8.0) and repeated a third time with filter paper sheets saturatedwith 1.0 M Tris-HCl (pH 8.0) containing 1.5 M NaCl for purposes ofneutralization. The nitrocellulose filters treated in this manner werethen air dried and baked in vacuo at 80° C. for 2 hours.

Prior to hybridization these nitrocellulose filters were next treatedfor 6-18 hours by incubating at 60° C. in a prehybridization bufferwhich is an aqueous mixture of 6×SSC [1×SSC=0.15 M NaCl and 0.015 M Nacitrate (pH 7.2)], 1×Denharts [1×Denharts=a solution containing 0.2%each of Ficoll, BSA, and polyvinylpyrrolidone], and 100-200 μg ofdenaturated sheared salmon sperm DNA (S.S. DNA)/ml, 1 mM EDTA, and 0.1%SDS. This treatment will decrease the amount of binding between thefilter and non-hybridized probe DNA next to be applied to the filters.

To screen for recombinant plasmids containing HS TK inserts, thetransformed colonies fixed to the original, treated, nitrocellulosefilters were hybridized with ³² P labelled Bam HSV TK fragment byimmersion of the filters in hybridization buffer containing 2×SSC (pH7.2), 1× Denhart's solution, 50 μg of S.S. DNA/ml, 1 mM EDTA, 0.1% ofSDS, 10% of dextran sulfate, and ³² P Bam TK as the hybridization probe.The level of radioactivity of the solution was approximately 100,000counts per minute (cpm) per milliliter.

Hybridization was effected at 60° C. over 18-24 hours [Wahl et al. Proc.Natl. Acad. Sci. USA 76, 3683-3687 (1979)].

[To prepare the hybridization probe, the 2.3 md Bam TK fragment waslabelled by nick translation according to the method of Rigby et al., J.Mol. Biol. 113, 237-251 (1977). More specifically, 0.1 ml of a reactionmixture was prepared containing 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 20uM deoxycytidine triphosphate (dCTP), 20 μM deoxyadenosine triphosphate(dATP), 20 μM deoxyguanosine triphosphate (dGTP), 2 μM (alpha-³²P)deoxythymidine triphosphate (dTTP) (410 Curies/m mol) (AmershamCorporation), 1 ng of DNase I, 100 units of DNA polymerase I (BoehringerMannheim), and 1 μg of Bam TK fragment. The reaction mixture wasincubated at 14° C. for 2 hours. The reaction was terminated by adding50 ul of 0.5 M EDTA and heating to 65° C. for 10 minutes. Unincorporatedtriphosphates were removed by gel filtration of the reaction mixture onSephadex G50.]

After hybridization, excess probe was removed from the nitrocellulosefilters by washing 5 times in 2×SSC (pH 7.2) containing 0.1% of SDS atroom temperature, followed by 3 washes in 0.2×SSC (pH 7.2) containing0.1% of SDS at 60° C., with each wash lasting 30 minutes. The washedfilters were then air dried and used to expose X-ray film (Kodak X-omatR) at -70° C. for 6-18 hours using a Cronex Lightening Plus intensifyingscreen (du Pont) for enhancement.

The exposed and developed X-ray film was then used to determine whichcolonies contained pBR 322 vaccinia Bam HSV TK recombinant plasmids.Those colonies which exposed the X-ray film were located on thecorresponding replica nitrocellulose filter. Such positive colonies werthen picked from the replica filters for further analysis. Of theapproximately 1000 colonies screened in this manner, 65 colonies weretentatively identified as having a Bam TK insert within pDP 3.

EXAMPLE VII Restriction Analysis of Recombinant Plasmids Containing BamHSV TK

Each of the 65 colonies which were tentatively identified as containingrecombinant plasmids with Bam HSV TK inserts were used to inoculate 2.0ml cultures of LB broth containing ampicillin at 100 μg/ml. The cultureswere then incubated at 37° C. overnight. Plasmids were extracted fromeach culture as described in Example III. The plasmids were dissolved ina 50 μl of water after isopropanol precipitation.

To determine if the plasmids contained an intact 2.3 md Bam HSV TKfragment and at which Bam HI site within pDP 3 the Bam HSV TK wasinserted, 25 μl of each plasmid preparation were mixed with 25 μl of2×Hind III buffer and digested at 37° C. for 2 hours with 1 unit of HindIII. The resulting fragments were then analyzed by electrophoresis on a1.0% agarose gel as described previously.

Of the 65 plasmid preparations analyzed 6 were found to contain Bam HSVTK fragments inserted into the Bam HI site present in the vaccinia HindIII F portion of the plasmid, i.e. they yielded Hind III restrictionfragments of molecular weights corresponding to linear pBR 322 (2.8 md)and fragments of a molecular weight greater than that of the vacciniaHind III F fragment (8.6 md).

These 6 plasmids were further analyzed with Sst I (an isoschizomer ofSac 1) to determine the number and orientation of the Bam HSV TKfragments inserted within vaccinia Hind III F Fragment, since Sst I (SacI) cleaves both the Bam HSV TK fragment and the vaccinia Hind III Ffragment asymetrically. The analyses were performed by mixing 25 μl ofthe plasmid with 25 μl of 2×Sst buffer [50 mM Tris-HCl (pH 8.0), 10 mMof MgCl₂, 100 mM of NaCl, and 10 mM of DTT] and digesting with 1 unit ofSst I (Bethesda Research Laboratories ) at 37° C. for 2 hours. Theresulting fragments were analyzed by electrophoresis in 1% agarose gels.Of the 6 plasmids analyzed, 5 yielded two Sst I fragments with molecularweights of 10.1 md and 3.5 md, indicating a single Bam HSV TK insert.One of these plasmids was selected for further study and designated pDP132. The other plasmid yielded three Sst I fragments with molecularweights of 10.8 md, 2.8 md, and 2.3 md, indicating tandom Bam HSV TKinserts oriented head to tail and in the opposite orientation ascompared to pDP 132. This plasmid was designated pDP 137. The plasmidspDP 132 and pDP 137 are diagramed in FIG. 3C.

EXAMPLE VIII Isolation of a TK⁻ S-variant Vaccinia Virus

To isolate a TK⁻ S-variant vaccinia virus mutant, a virus population wassubjected to strong selective pressure for such a mutant by growing thevirus in cells in the presence of BUdR, which is lethal to organismscarrying the TK gene. More in particular, confluent monolayers of TK⁻human (line 143) cells growing in Eagle's Special medium in 150 mm Petridishes were infected with approximately 3×10³ plaque forming units (pfu)of S-variant vaccinia virus per dish (20 dishes used) in the presence of20 ug BUdR/ml. (Eagle's Special medium is a commercially availablenutrient medium for the growth of most cell lines. Alternative mediasuch as Eagle's Minimum Essential Medium, Basal Eagle's Medium,Ham's-F10, Medium 199, RPMI-1640, etc., could also be used.) Growth isat 37° C. in an atmosphere enriched in CO₂. This is conveniently doneusing a CO₂ -incubator providing air enriched with CO₂ to have a CO₂content of about 5 percent.

Ninety-three of the plaques which developed were isolated and replaquedon TK⁻ human (line 143) cells under the conditions mentioned previouslyand again in the presence of 20 μg of BUdR/ml. A number (5) of large,well isolated plaques were picked for further analysis.

The five plaque isolates were tested for growth on cell monolayers underthe same conditions used earlier and in the presence or absence of 20 ugBUdR/ml. The relative growth of each plaque in the presence and absenceof BUdR was noted and compared with the relative growth in similarmonolayer cell cultures of the parent S-variant virus. The followingresults were obtained:

    ______________________________________                                        Plaque Isolate                                                                             -BUdR (pfu/ml)                                                                             +BUdR (pfu/ml)                                      ______________________________________                                        #70          5.1 × 10.sup.5                                                                       4.1 × 10.sup.5                                #73          1.0 × 10.sup.6                                                                       1.0 × 10.sup.6                                #76          4.7 × 10.sup.5                                                                       4.7 × 10.sup.5                                #79          5.4 × 10.sup.5                                                                       4.4 × 10.sup.5                                #89          5.9 × 10.sup.5                                                                       7.0 × 10.sup.5                                S-variant    .sup. 1.7 × 10.sup.10                                                                9.7 × 10.sup.6                                ______________________________________                                    

The growth of plaque isolate #79 was further monitored in the presenceof 0, 20 and 40 ug BUdR/ml and compared with the growth of its parentS-variant virus. The following results were obtained:

    ______________________________________                                        Yield (pfu/ml)                                                                Virus    0 μg/ml    20 μg/ml                                                                            40 μg/ml                                   ______________________________________                                        #79      2.5 × 10.sup.5                                                                        4.l × 10.sup.5                                                                   3.2 × 10.sup.5                          S-Variant                                                                              1.2 × 10.sup.9                                                                        1.3 × 10.sup.6                                                                   2.0 × 10.sup.5                          ______________________________________                                    

In addition, the above 5 plaque isolates and the S-variant parent weremonitored for growth on TK⁻ human (line 143) cells in the presence ofMTAGG. MTAGG is an Eagles's Special medium modified by the presence of:

    ______________________________________                                        8 × 10.sup.-7 M                                                                              methotrexate                                             1.6 × 10.sup.-5 M                                                                            thymidine                                                5 × 10.sup.-5 M                                                                              adenosine                                                5 × 10.sup.-5 M                                                                              guanosine                                                1 × 10.sup.-4 M                                                                              glycine                                                  ______________________________________                                    

(cf. Davis et al., op. cit.) and selects for thymidine kinase andagainst organisms free of the thymidine kinase gene. The results of suchan experiment were as follows:

    ______________________________________                                                    Plaque Forming Units/ml                                           Virus         -MTAGG    +MTAGG                                                ______________________________________                                        #70           4.0 × 10.sup.5                                                                    0                                                     #73           5.8 × 10.sup.5                                                                    0                                                     #76           2.8 × 10.sup.5                                                                    3.3 × 10.sup.3                                  #79           3.6 × 10.sup.5                                                                    0                                                     #80           4.3 × 10.sup.5                                                                    4.0 × 10.sup.3                                  S-Variant     4.8 × 10.sup.9                                                                    2.6 × 10.sup.9                                  ______________________________________                                    

Of the three plaque isolates showing complete inhibition of growth inthe presence of MTAGG, isolate #79 was arbitrarily selected and extractsprepared from cells infected with #79 virus were compared with extractsprepared from uninfected cells and from cells infected with theS-variant parent virus with respect to the ability of the extracts tophosphorylate tritiated (³ H) thymidine. The results are tabulatedbelow:

    ______________________________________                                                         .sup.3 H Thymidine                                                            Phosphorylated                                               Extract Source   (cpm/15 μg Protein)                                       ______________________________________                                        Uninfected TK.sup.- human                                                                         0                                                         (line 143)                                                                    #79 infected cells                                                                                90                                                        S-variant infected cells                                                                       66,792                                                       ______________________________________                                    

In view of (1) resistance to BUdR, (2) inhibition of growth by a mediumcontaining MTAGG, and (3) failure to detect significant phosphorylationof thymidine in infected cell extracts, plaque isolate #79 is consideredto lack thymidine kinase activity. The isolate is designated VTK⁻ 79.

EXAMPLE IX Marker Rescue of L-variant Vaccinia DNA by the S-Variant

Four preparations of L-variant DNA were prepared for marker rescuestudies. The first consisted of purified, intact, L-variant vacciniaDNA. The second consisted of L-variant vaccinia DNA digested with Bst EII, a restriction endonuclease which generates a donor DNA fragment,fragment C, comprising that DNA which is absent from the S-variant anduniquely present in the L-variant and which also has, at both ends ofthe DNA chain, a region of DNA homologous with corresponding sequencesin the S-variant. The third and fourth preparations respectivelyconsisted of L-variant DNA digested with Ava I and Hind III, restrictionendonucleases that cleave the vaccinia genome within the uniqueL-variant DNA sequence. The marker rescue studies performed with thesefour preparations demonstrate that those L-variant DNA fragmentscontaining the deleted region absent from the S-variant can bereintroduced into the S-variant by an in vivo recombination techniqueproviding that the fragment contains, in addition to the deleted region,terminal regions which are homologous with corresponding sequences inthe S-variant.

A better understanding of the fragments employed in these studies willbe had by referring to FIGS. 6 A-C, each of which is a restriction mapof a portion of the left terminus of the vaccinia genome. More inparticular, each map refers to the left-terminal region of the genomecomprising approximately 60 kilobasepairs, as indicated in the Figure.The portion of the vaccinia genome which is deleted from the S-variantis represented in each map as the region between the dotted lines shownin the Figures, a region approximately 10 kilobasepairs in length.

Turning now more specifically to FIG. 6A, it is evident that fragment Hobtained by digestion with Ava I is completely within the deleted regionbut will have no terminal DNA fragments homologous with the DNA of theS-variant because the Ava I cleavage sites fall entirely within thedeleted region of the S-variant.

The restriction map of FIG. 6B pertaining to Hind III shows that thisrestriction enzyme similarly fails to produce a L-variant DNA fragmentoverlapping the deleted region of the S-variant. In this instance,sequences homologous with the S-variant are found at the left terminusof the C-fragment of Hind III. However, the restriction site at theright-hand terminus of fragment C falls within the deleted region andthere is no terminal sequence homologous with the DNA sequence of theS-variant.

In contrast, the restriction map shown in FIG. 6C pertaining to Bst E IIshows that digestion with this enzyme produces a fragment, fragment C,which includes the deleted region absent from the S-variant and also hasterminal portions at both the left and right ends which are homologouswith corresponding portions of the S-variant.

The results of the experiments, discussed more in detail below, indicatethat the DNA which is present in the L-variant but is deleted from theS-variant is rescued by the S-variant with high efficiency from theintact L-variant genome, is rescued with lower efficiency from the Cfragment of Bst E II, and cannot be rescued from either of the L-variantDNA fragments prepared with the Ava I and Hind III restrictionendonucleases.

The high efficiency with which the deleted sequence is rescued from theintact L-variant is attributable to the fact that a single crossoverbetween the intact L-variant and the S-variant is sufficient to producean L-variant genome type. On the other hand, to rescue the deletedportion from the C fragment of Bst E II, a crossover between thefragment and the S-variant is necessary in both the left- and right-handterminal portions of the C-fragment in order to incorporate the deletedregion into the S-variant. Finally, since neither digestion with Ava Inor with Hind III produces DNA fragments which can be incorporated intothe S-variant by any crossover, no rescue of the deleted portion iseffected.

The marker rescue was performed on CV-1 monolayers using the calciumphosphate technique of Graham et al., Virology, 52, 456-467 (1973), asmodified by Stow et al. and Wigler et al., both mentioned earlierherein. Confluent CV-1 monolayers were infected with S-variant vacciniavirus to give approximately 50 to 200 plaques in each of a number (5-20)of Petri-dishes of 6 cm diameter. To infect the cells, the growth medium(e.g. Eagle's Special containing 10% calf serum) is aspirated and adilution of the virus containing 50-200 pfu/0.2 ml in a cell-compatiblemedium such as Eagle's Special containing 2% calf serum is applied tothe cell monolayer. After incubation for a period of one hour at 37° C.in a CO₂ -incubator to permit the absorption of the virus to the cells,various of the four L-variant DNA preparations earlier mentioned wereeach separately added to the monolayers as a calcium phosphateprecipitate containing one microgram per dish of the L-variant DNApreparation. After 40 minutes, Eagle's Special medium with 10% calfserum was added and, four hours after the initial addition of the DNA,the cell monolayer was exposed to 1 ml of buffered 25 percent dimethylsulfoxide for four minutes. This buffer contains 8 g of NaCl, 0.37 g ofKCl, 0.125 g of Na₂ HPO₄.2H₂ O, 1 g of dextrose, and 5 g ofN-(2-hydroxyethyl)piperazine,N'-(2-ethanesulfonic acid) (Hepes) perliter, having a final pH of 7.05. The dimethylsulfoxide was removed andthe monolayers were washed and overlayed with nutrient agar. After threedays, at 37° C. in a CO₂ -incubator, the cells were stained with anutrient agar overlay containing Neutral red dye, which stains theuninfected cells (nutrient agar=Eagle's Special medium containing 10%calf serum and 1% agar). The next day, the agar overlay was removed andthe monolayers were transferred to nitrocellulose filters and preparedfor in situ hybridization as described by Villarreal et al., loc. cit.Since digestion of the L-variant genome with Ava I generates a 6.8kilobasepair fragment, fragment H, that resides entirely with the uniqueDNA sequences deleted in the S-variant genome (cf. FIG. 6A), ³²P-labelled nick-translated Ava I H fragment provides a highly specificprobe for detecting the rescue of the unique L-variant DNA sequence bythe S-variant.

For hybridization, the nitrocellulose filters were interleaved withWhatman No. 1 filter paper circles in 6 cm Petri dishes and werprehybridized for 6 hours at 60° C. in prehybridization buffer (SSCDenhardt solution, EDTA, and S.S.DNA) as described earlier herein inExample VI. The radioactive probe consisting of ³² P-labellednick-translated L-variant Ava I, H fragment, having a specific activityof approximately 1×10⁸ cpm/μg was used for hybridization in 2×SSC,1×Denhardt, 1 mM EDTA, 0.1 percent SDS, 10 percent of dextran sulfate,and 50 μg/ml of sonicated S.S.DNA at approximately 1×10⁵ cpm/mlovernight at 60° C. The radioactive probe was prepared according to themethod of Rigby et al., J. Mol. Biol. 113, 237-251 (1977). The filterswere washed repeatedly at room temperature and at 60° C. using thewashing procedure of Example VI, were air dried, and radioautographed.

The results of the experiments are summarized in Table I below:

                  TABLE I                                                         ______________________________________                                                       Percent of Plaques                                             Donor L-variant                                                                              Containing L-variant                                           DNA Preparation                                                                              Genotype                                                       ______________________________________                                        Intact L-variant                                                                             5                                                              Bst E II total digest                                                                        0.1                                                            Ava I total digest                                                                           0                                                              Hind III total digest                                                                        0                                                              ______________________________________                                    

A minimum of 5000 plaques were analyzed for each donor DN preparation.

EXAMPLE X In vivo Recombination Using pDP 132 and pDP 137 to GenerateVaccinia Virus Mutants VP-1 through VP-6 and Identification ThereofUsing Replica Filters

A first calcium orthophosphate precipitate of donor DNA was prepared bycombining 5 μg of pDP 132 Hind III digested DNA in 50 μl of water, 4 μgof S-variant carrier DNA (prepared as in Example I) in 40 μl of water,and 10 μl of 2.5M CaCl₂, combining the resultant mixture with an equalvolume of 2×Hepes phosphate buffer comprising 280 mM NaCl, 50 mM Hepes,and 1.5 mM sodium phosphate (pH=7.1), and permitting the precipitate toform over a period of 30 minutes at room temperature. A secondprecipitate was prepared in the same fashion, in the same amounts, butusing pDP 137 Hind III digested DNA.

[As described more in detail by Stow et al. loc. cit. and Wigler et al.,loc. cit., the modifications of the Graham et al. precipitationtechnique referred to earlier employ carrier DNA as high molecularweight substance increasing the efficiency of calcium orthophosphateprecipitate formation. The carrier DNA employed is DNA from the viruswhich is used for infection of the monolayered cells in the in vivorecombination technique.]

For in vivo recombination, confluent monolayers of CV-1 growing inEagle's Special medium containing 10% calf serum were infected withS-variant vaccinia virus at a multiplicity of infection of 1 pfu/cell.The infection procedure is like that described in Example IX. The viruswas permitted to absorb for 60 minutes at 37° C. in a CO₂ -incubator,after which the innoculum was aspirated and the cell monolayer waswashed. The precipitated DNA preparations were applied to separate cellmonolayers and, after 40 minutes at 37° C. in a CO₂ -incubator, liquidoverlay medium was added (Eagle's Special containing 10% calf serum). Ineach case, the virus was harvested after 24 hours at 37° C. in a CO₂-incubator by 3 freeze/thaw cycles and titered on CV-1 monolayers.Approximately 15,000 plaques were analyzed on CV-1 monolayers forrecombinant virus using replica filters prepared as follows.

Plaques formed on confluent CV-1 monolayers under a nutrient agaroverlay were transferred to a nitrocellulose filter by removing the agaroverlay cleanly with a scalpel and placing the nitrocellulose filteronto the monolayer. Good contact between the filter and monolayer waseffected by placing a Whatman No. 3 filter paper, wetted in 50 mM Trisbuffer (pH=7.4) and 0.015 mM NaCl over the nitrocellulose filter andtamping with a rubber stopper until the monolayer transferred to thenitrocellulose shows a uniform color surrounding discrete uncoloredplaques. (The monolayer has been previously stained with Neutral red,which is taken up by viable cells, i.e. cells unlysed by virusinfection).

The nitrocellulose filter having the transferred monolayer thereon isnow removed from the Petri dish and placed with the monolayer side up. Asecond nitrocellulose filter, wetted in the above-mentioned Tris-NaClsolution, is now placed directly over the first nitrocellulose filterand the two filters are brought firmly into contact by tamping with arubber stopper after protecting the filters with a dry Whatman No. 3circle. After removing the filter paper, the nitrocellulose filters arenotched for orientation and separated. The second (replica)nitrocellulose filter now contains a mirror image of the cell monolayertransferred to the first nitrocellulose filter. The second filter isconveniently placed in a clean Petri dish and frozen. The firstnitrocellulose filter is subjected to hybridization employing ³²P-labelled Bam HSV TK fragment as a probe. The preparation of the probeand the hybridization technique are described earlier herein in ExampleVI.

Approximately 0.5 percent of the plaques analyzed by hybridization werepositive, i.e. were recombinant virus containing Bam HSV TK.

Plaques of recombinant virus corresponding to those identified on thefirst nitrocellulose filter by hybridization were then isolated from thenitrocellulose replica filter by the following technique for furtherpurification.

Using a sharp cork borer having a diameter slightly larger than theplaque to be picked, a desired plaque is punched out from the first ororiginal nitrocellulose filter which has been used for identification ofrecombinants by hybridization. The resulting perforated filter is nextused as stencil to identify and isolate the corresponding plaque on thereplica filter. Namely, the replica filter is placed with the monolayerside up on a sterile surface and covered with a sheet of Saran wrap. Theperforated first or original nitrocellulose filter is then placedmonolayer side down over the second filter and the orientation notchespresent in the filters are aligned to bring the mirror images of theplaques into register. Again, using a cork borer, a plug is removed fromthe replica filter and, after removal of the Saran wrap protectivelayer, is placed in one ml of Eagle's Special medium containing 2% calfserum. The nitrocellulose plug is sonicated in this medium for 30seconds on ice to release the virus. 0.2 ml of this virus preparation,and 0.2 ml of a 1:10 dilution of the preparation, are plated on CV-1monolayers present in 6 cm Petri dishes.

As a plaque purification step, the entire sequence of preparing a firstnitrocellulose filter, a replica filter, hybridization, and plaqueisolation from the replica filter was repeated.

One sample of a purified plaque prepared in this manner starting from acalcium orthophosphate precipitate of pDP 132 Hind III digested DNA wasdenominated vaccinia virus VP-1. Similarly, a plaque containing arecombinant prepared from pDP-137 Hind III digested DNA was denominatedVP-2. Both samples were grown up on suitable cell cultures for furtherstudy, including identification by restriction analysis and othertechniques.

In like fashion, two further vaccinia mutants respectively denominatedVP-3 and VP-4 were prepared by in vivo recombination employing VTK-79(an S-variant TK⁻ vaccinia virus as described in Example VIII) as therescuing virus and, respectively, pDP 132 and pDP 137 as the plasmiddonor DNA. The precipitates were formed as described earlier hereinexcept that 5 μg of plasmid donor DNA present in 50 μl of water, 4 μg ofVTK⁻ 79 carrier DNA in 150 μl of water, and 50 μl of 2.5M CaCl₂ werecombined and added dropwise to an equal volume of 250 μl of the Hepesphosphate buffer earlier described.

Further, the cells employed for infection by the VTK⁻ 79 virus carrierwere BHK-21 (Clone 13) cells instead of CV-1.

Two further vaccinia virus mutants denominated VP-5 and VP-6 wereprepared using calcium orthophosphate precipitates of pDP 132 and pDP137, respectively, each as prepared for mutants VP-3 and VP-4. However,in the case of mutants VP-5 and VP-6, the carrier DNA is vaccinia virusVTK⁻ 11, rather than VTK ⁻ 79.

Again, BHK-21 (C-13) cell monolayers were infected, the rescuing virusin this case being VTK⁻ 11.

EXAMPLE XI Expression of HSV TK by Vaccinia Mutant VP-2 and the Use ofIDC* for Identification Thereof

The virus product obtained in Example X by the in vivo recombination ofS-variant vaccinia virus and the calcium orthophosphate precipitate ofpDP-137 Hind II digested DNA was plated out on confluent monolayers ofCV-1 cells present on approximately twenty 6 cm Petri dishes at aconcentration giving approximately 150 plaques per dish. The plaqueswere covered with a liquid overlay medium, e.g. Eagle's Special mediumcontaining 10% calf serum. After 24 to 48 hours of incubation at 37° C.in a CO₂ -incubator, the liquid overlay medium was removed from thedishes and replenished in each case with 1.5 ml of the same liquidoverlay medium containing 1-10 μCi of ¹²⁵ I iododeoxycytidine (IDC*).The plates were then further incubated overnight, at 37° C. in anenriched CO₂ atmosphere, after which the cell monolayer present thereonwas stained by the addition of Neutral red to visualize the plaques bycontrast.

The medium was then removed by aspiration, the monolayers were washedthree times with phosphate-buffered saline solution, and the cellmonolayer on each of the plates was imprinted onto a correspondingnitrocellulose filter. The latter was exposed to X-ray film for from 1to 3 days and then developed.

Those viral plaques containing and expressing the HSV TK gene willphosphorylate IDC* and incorporate it into their DNA, rendering the DNAinsoluble. Other, unphosphorylated and unincorporated, IDC* was removedby washing, so that plaques darkening the X-ray film are thoseexpressing recombinant HSV TK gene. Neither CV-1 cells nor vaccinia,although containing TK, will phosphorylate and incorporate IDC* in theselective fashion characteristic of the HSV TK.

After the recombinant organisms has been identified by radioautography,filter plugs were cut from the nitrocellulose filter, placed in 1 ml ofoverlay medium, (Eagle's Special, 10% calf serum), sonicated, andreplated on CV-1 monolayers. The IDC* assay was then repeated further topurify the viral isolates. In this manner, a virus identical to the VP-2mutant identified by hybridization in Example X was isolated by atechnique dependent on the expression of the HSV TK gene presenttherein.

Again, the results of this Example demonstrate the expression of the HSVTK gene, present in the recombinant organisms according to the presentinvention by certain of those organisms.

Those vaccinia mutants derived from pDP 137, namely VP-2, VP-4, andVP-6, all will express the HSV TK gene present therein byphosphorylation and incorporation of IDC* in the manner described above.However, the variants VP-1, VP-3, and VP-5, derived from pDP 132, willnot so express the gene, possibly because the orientation of the genewithin the virus is contrary to the direction of gene transcription.

EXAMPLE XII The use of a Selective Medium for the Identification andIsolation of Recombinant Virus Containing HSV TK Gene

Viruses prepared according to Example X by the in vivo recombination, inBHK-21 (C-13) cells, of VTK⁻ 79 vaccinia virus and a calciumorthophosphate precipitate of pDP 137 were used to infect human (line143) TK⁻ cells. More in particular, cell monolayers, in five Petridishes 6 cm in diameter, were each infected with the virus of Example Xat a dilution of the virus from 10⁰ to 10⁻⁴ in the presence of selectiveMTAGG medium. The infection technique was as described earlier.

Five well-separated plaques were isolated and one was replated on CV-1monolayers for a second cycle of plaque purification. One furtherwell-separated plaque, purified twice by plaque purification, was chosenand analyzed. A well-isolated plaque, thus twice plaque-purified, wasselected and analyzed for the presence of the HSV TK gene by in situhybridization employing ³² P-labelled Bam HSV TK. The hybridizationtechnique was, again, as described earlier. The mutant vaccinia virus,positive for the presence of the HSV TK gene, was denominated VP-4.

What is claimed is:
 1. A method for the in vivo recombination ofvaccinia virus DNA with DNA not naturally occurring in vaccinia virus,which method comprises infecting a cell with vaccinia virus in acell-compatible medium in the presence of doner DNA, said donor DNAcomprising said DNA not naturally occurring in vaccinia virus flanked byDNA sequences homologous with portions of the vaccinia genome, wherebysaid DNA not naturally occurring in vaccinia is introduced into thegenome of said vaccinia virus, and then recovering vaccinia virusmodified by such in vivo recombination.
 2. A method as in claim 1wherein said flanking DNA sequences are co-linear with portions of thegenome of the vaccinia virus except for the presence of said DNA notnaturally occurring in vaccinia virus.
 3. A method as in claim 1 whereinsaid flanking sequences are co-linear with portions of the genome of thevaccinia virus in a non-essential region thereof, except for thepresence of said DNA not naturally occurring in vaccinia virus.
 4. Amethod for the in vivo recombination of vaccinia virus DNA and DNA notnaturally occurring in vaccinia virus, which method comprises infectinga cell with vaccinia virus in a cell-compatible medium in the presenceof donor DNA, said donor DNA comprising said DNA not naturally occurringin vaccinia virus present within a segment of vaccinia DNA, whereby saidDNA not naturally occurring in vaccinia is introduced into the genome ofsaid vaccinia virus, and then recovering vaccinia virus modified by suchin vivo recombination.
 5. A method as in claim 4 wherein said segment ofvaccinia DNA is co-linear with portions of the vaccinia genome exceptfor the presence of said DNA not naturally occurring in vaccinia virus.6. A method as in claim 5 wherein said DNA not naturally occurring invaccinia virus is present within a non-essential region of said segmentof vaccinia DNA.
 7. A method as in claim 5 wherein said cell is presentin a cell monolayer.
 8. A method for the in vivo recombination ofvaccinia virus DNA with DNA not naturally occurring in vaccinia virus,which method comprises contacting a cell monolayer with acell-compatible medium containing vaccinia virus and with acell-compatible medium comprising donor DNA, said donor DNA comprisingsaid DNA not naturally occurring in vaccinia virus present within anon-essential region of a segment of vaccinia virus otherwise co-linearwith portions of the vaccinia genome, whereby said DNA not naturallyoccurring in vaccinia is introduced into the genome of said vacciniavirus, and then recovering vaccinia virus modified by such in vivorecombination.
 9. A method as in claim 8 wherein said donor DNA isclonable with a plasmid.
 10. A method as in claim 8 wherein said DNA notnaturally occurring in vaccinia virus comprises the Bam HI TK gene ofherpes simplex.
 11. A method as in claim 8 wherein said co-linearsegment of vaccinia DNA is the Hind III F-fragment thereof.
 12. A methodas in claim 8 wherein said donor DNA is present as a calciumorthophosphate precipitate of said donor DNA.
 13. A method as in claim 8wherein said vaccinia virus is free of a vaccinia gene producingthymidine kinase.
 14. The method of replicating DNA in a eukaryotic cellby infecting said cell with a vaccinia virus modified to contain saidDNA, said DNA not naturally occurring in vaccinia virus.
 15. A method asin claim 14 wherein said DNA is also not naturally occurring in saidcell.
 16. A method as in claim 14 wherein said DNA is expressed by saidcell.
 17. A method as in claim 15 wherein said DNA is expressed by saidcell.
 18. A method as in claim 16 wherein said DNA is expressed by theproduction by said cell of a biologically active protein.
 19. A methodas in claim 17 wherein said DNA is expressed by the production by saidcell of a biolgically active protein.